Defects in the Hedgehog (Hh) pathway underlie a vast spectrum of human birth defects, including holoprosencephaly, cleft palate, and polydactyly. Hh signaling controls transcription by regulating the activity of Gli transcription factors. A thorough understanding of how Gli proteins recognize and activate transcriptional targets is therefore necessary to understand the genes and processes that are misregulated in Hh-associated birth defects. However, virtually nothing is known about how Gli proteins recognize target genes and interact with other transcriptional inputs. The aim of this proposal is to define active Gli enhancers and to determine their interactions in a Hh- responsive cis regulatory network that underlies limb development. In the first approach, we will use an innovative approach to identify Hh-responsive, active enhancers different genetic backgrounds. This data will be used to construct a global gene regulatory network, defining an important set of extragenic regions with the collective potential to mediate the transcriptional effects of the Hh pathway. In a second in vivo-centered approach, we will determine the requirement for additional co-activating factors in the transcriptional regulation of Hh target genes. Finally, we will determine if the Hh and BMP pathways co-regulate target genes, focusing on the regulation the developmentally important gene, Gremlin. This study will transform our understanding of limb development by identifying active Gli enhancers and target genes in the limb, defining enhancer domains and determining co-factors. These will provide an invaluable resource for future studies and will provide a solid foundation for a systems-level understanding of limb development. The identification of these enhancers will also provide an important genomic resource for clinical scientists seeking to understand the mechanisms underlying the spectrum of BMP and Hh-associated birth defects and the hundreds of clinically distinct forms of human polydactyly.